This invention concerns generally the purification of a therapeutically useful protein and specifically the purification to near homogeneity of alpha-1 proteinase inhibitor (xcex11-PI), especially transgenic human xcex11-PI from a non-human animal source.
Currently, xcex11-PI (also known as xcex11-antitrypsin inhibitor) derived from human plasma is commercially available (Prolastin(copyright) xcex11-PI, Bayer Corporation) to treat congenital deficiencies of the protein. Such plasma-derived xcex11-PI comprises about 85% xcex11-PI of total protein on a wt/wt basis. Human plasma as a source, however, has disadvantages, including a limited supply, and the potential of viral contamination. These disadvantages have prompted investigations into a variety of recombinant sources so that the existing patient population could be more fully treated and the number of indications expanded without the above disadvantages.
Recombinant human xcex11-PI has been produced in both E. coli and yeast (Courtney et al., 1984; Sleep et al., 1991), but the lack of post-translation glycosylation by the micro-organisms has led to unacceptably high pharmacokinetic clearance rates. While the conventional solution to this problem is to tansfect mammalian cells to make a recombinant form of the protein, this approach is too costly given the large dose of xcex11-PI needed for congenitally deficient patients (60 mg/kg/week).
Wright et al., 1991, describe transgenic sheep that express human xcex11-PI with mammalian-like glycosylation in their milk at up to 30 g/l. Wright et al. (1994), without giving details, further report having isolated xcex11-PI xe2x80x9cof very high purity ( greater than 99%)xe2x80x9d. However, our initial studies demonstrated that the major obstacles in preparing a pharmaceutical grade preparation of this protein for clinical use would be in effectively eliminating the remaining sheep whey protein. A particular problem involved selectively eliminating sheep xcex11-PI from the transgenic human xcex11-PI.
The prior art reveals a number of methods which have been used to purify xcex11-PI. Bischoff et al. (1991) have isolated site-directed mutant xcex11-PI produced by E. coli. Their method consisted of chromatography over a silica-based anion exchange substrate, zinc-chelate chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography. The resultant product was reported to be xe2x80x9cof high purity as determined by PAGE under reducing conditions in the presence of SDSxe2x80x9d (pp. 3468-3469). HPLC analysis of the product showed no contaminant at greater than 1%, the detection limit of the instrumentation.
Hoylaerts et al. (1986) have used a small scale immunoaffinity chromatography process to obtain non-glycosylated recombinant xcex11-PI which was about 80% pure. They also developed a large scale purification process for xcex11-PI which consisted of precipitation with poly(ethylene glycol), DEAE-Sepharose(copyright) chromatography, zinc-chelate chromatography, a kappa-chain-agarose chromatography step, a heparin-agarose chromatography step, and an aminohexyl-agarose chromatography step. The six step process yielded material which was greater than 95% pure.
However, the xcex11-PI isolated by Bischoff et al. and Hoylaerts et al. is non-glycosylated and therefore is of limited pharmacological use. Archibald et al. (1990) report characterization of transgenic human xcex11-PI produced in the milk of transgenic mice. Mistry et al. (1991) have developed a purification process for native, glycosylated sheep xcex11-PI which consists of ammonium sulfate precipitation, concanavalin A chromatography, anion exchange chromatography on a Mono Q(copyright) column, and preparative scale native-PAGE. A further step used was immunoadsorbant column chromatography to obtain a product which was greater than 98% pure as measured by SDS-PAGE.
However, even a purity of 98+% would be inadequate for parenteral drug use of xcex11-PI isolated from a non-human source. The potential immune response to contaminants necessitates a product of extremely high purity. It is thus of primary importance to discover a method which results in a product of the requisite purity for parenteral use.
Such a high level of purity requires a multistep process, with a high yield at each step. The prior art reveals anion exchange chromatography steps, fractionation steps using either poly(ethylene glycol) or ammonium sulfate, and immunoaffinity chromatography steps. Bollen et al.(1986) report a multistep process for purifying xcex11-PI which yields xe2x80x9chighly purifiedxe2x80x9d xcex11-PI with xe2x80x9ctrace contaminantsxe2x80x9d. The Bollen process includes anion exchange, thiol exchange, heparin-affinity, and Zn-chelate chromatography steps.
The use of an immobilized zinc affinity chromatography step for purification of xcex11-PI is detailed in the prior art. See, for example, Kurecki, et al., 1979. The use of cation exchange to purify a complex of native human xcex11-PI and proteinase-3 has been reported by Ballieux et al. (1993). The complex isolated by Ballieux et al. is too small to contain intact proteins, and is most likely a degradation product of the complexed proteins. The use of a cation exchange chromatography step in a scheme for purifying intact, native human xcex11-PI derived from plasma also was described by Lebing and Chen (1994).
We have prepared an essentially homogenous transgenic human alpha-1 proteinase inhibitor (tg xcex11-PI) which has a purity substantially greater than 99.99 g tg xcex11-PI/100 g total protein. This product is preferably prepared by subjecting a solution containing impure tg xcex11-PI to a series of chromatography steps comprising at least one cation exchange step. The preferred embodiment includes anion exchange, protein G affinity chromatography, immobilized nickel affinity chromatography, and hydrophobic interaction chromatography steps. Preferably, the contact with the cation exchange material (resin) is performed at about pH 5.5 with the salt concentration at or below about 10 mM. In the most preferred embodiment, the purified tg xcex11-PI includes less than 40 pg of protein other than casein, native sheep xcex11-PI, and tg xcex11-PI per mg total protein.